This application claims benefit to Provisional Ser. No. 60/223,044 filed Aug. 4, 2000.
This invention relates to a process for the preparation of a 3-hydroxycarboxylic acid from a 3-hydroxynitrile. More particularly, 3-hydroxyvaleronitrile is converted to 3-hydroxyvaleric acid in high yield at 100% conversion, using as catalyst the nitrile hydratase and amidase activities, or the nitrilase activity, of a microbial cell. 3-Hydroxyvaleric acid is used as a substitute for xcex5-caprolactone in the preparation of highly branched polyesters.
Precise macromolecular engineering is becoming a major trend in polymer technology to satisfy the demand for new properties, improved cost effectiveness, ecology, and quality. Functional polymers with branched, compact structures and terminally located reactive groups are expected to exhibit superior performance/cost characteristics, by virtue of their lower inherent viscosity and higher reactivity versus conventional linear statistical copolymers. Preparation of these polymers can be accomplished by copolymerizing hyperbranching hydroxycarboxylic acid comonomers (hyperbranching ABn type, where A and B are moieties with hydroxyl- or carboxyl-derived reactive groups, n is 2 or more) (Hult et al., pp. 656-658 and Voit et al., pp. 658-659 in Concise Polymeric Materials Encyclopedia, ed. J. C. Salomone, CRC Press, New York, 1999) and a variety of linear hydroxycarboxylic acid comonomers (linear AB type), including 3-hydroxyvaleric acid.
3-Hydroxyvaleric acid is also useful as a (co)monomer for making linear polyesters. Polyesters are useful as thermoplastic, thermoset, semicrystalline, amphorous, rigid, and elastomeric materials. They are the basis of fibers, films, moldings, and coatings (Goodman, pp. 793-799 in Concise Encyclopedia of Polymer Science and Engineering, ed. J. I. Kroschwitz, John Wiley and Sons, New York, 1990).
3-Hydroxyvaleric acid has been prepared by the xcex2-hydroxylation of valeric acid in fermentation using Candida rugosa (Hasegawa et al., J. Ferment. Technol. 59:257 -262 (1981); JP 59053838 B4), and a single enantiomer of 3-hydroxyvaleric acid was similarly prepared by fermentative xcex2-hydroxylation of valeric acid with Pseudomonas putida, Pseudomonas fluorescens, Arthrobacter oxydans and Arthrobacter crystallopietes (U.S. Pat. No. 3,553,081). These methods for fermentative oxidation of valeric acid typically produce 3-hydroxyvaleric acid at low product concentrations, and require an elaborate and expensive separation of 3-hydroxyvaleric acid from the fermentation broth. (R)-(xe2x88x92)-3-Hydroxyvaleric acid has been prepared by the chemical degradation (Seebach et al., Helv. Chim. Acta 77:2007-2034 (1994)) or by fermentative autodegradation (WO 9929889) of poly(3-hydroxybutyrate/3-hydroxyvalerate), but degradation of hydroxybutyric acid/hydroxyvaleric acid copolymers also requires a difficult separation of 3-hydroxybutyric acid from the co-product 3-hydroxyvaleric acid. (R)-(xe2x88x92)-3-Hydroxyvaleric acid has also been prepared by the enzymatic reduction of 3-oxovaleric acid (Bayer et al., Appl. Microbiol. Biotechnol. 42:543-547 (1994)) or by the asymmetric hydrogenation of methyl 3-oxovalerate followed by saponification (Burk et al., Organometallics 9:2653-2655 (1990)).
Nitriles are readily converted to the corresponding carboxylic acids by a variety of chemical processes. These processes typically require strongly acidic or basic reaction conditions and high reaction temperatures, and usually produce unwanted byproducts and/or large amounts of inorganic salts as unwanted waste. Reaction conditions for the chemical hydrolysis of nitriles which additionally have a hydroxyl group, such as for the conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid, will usually also result in the undesirable elimination of primary, secondary, or tertiary hydroxyl groups to produce carbonxe2x80x94carbon double bonds.
The enzyme-catalyzed hydrolysis of nitriles substrates to the corresponding carboxylic acids is often preferred to chemical methods, since the reactions are often run at ambient temperature, do not require the use of strongly acidic or basic reaction conditions, and produce the desired product with high selectivity at high conversion.
A combination of two enzymes, nitrile hydratase and amidase, can be used to convert aliphatic nitrites to the corresponding carboxylic acid in aqueous solution. The aliphatic nitrile is initially converted to an amide by the nitrile hydratase, then the amide is subsequently converted by the amidase to the corresponding carboxylic acid. A wide variety of bacterial genera are known to possess a diverse spectrum of nitrile hydratase and amidase activities (Sugai et al., Biosci. Biotech. Biochem. 61:1419-1427 (1997)), including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium and Micrococcus. The fungus Fusarium merismoides TG-1 has also been used as catalyst for the hydrolysis of aliphatic nitrites and dinitriles (Asano et al., Agric. Biol. Chem. 44:2497-2498 (1980)). Immobilized nitrile hydratase and amidase from Rhodococcus sp. (SP409 from Novo Industri) was used to hydrolyze 3-hydroxypropionitrile, 3-hydroxyheptanenitrile, 3-hydroxyoctanenitrile, and 3-hydroxynonanenitrile to the corresponding 3-hydroxycarboxylic acids in 63%, 62% and 83% yields, respectively (de Raadt et al., J. Chem. Soc. Perkin Trans. 1, 137-140 (1992)). The formation of the corresponding amide was also observed by TLC. In contrast, the purified nitrile hydratase of Bacillus pallidus Dac521 hydrolyzed a variety of aliphatic nitrites, but did not hydrolyze 3-hydroxypropionitrile (Cramp et al., Biochim. Biophys. Acta 1431:249-260 (1999)).
A single enzyme, nitrilase, also converts a nitrile to the corresponding carboxylic acid and ammonia in aqueous solution, but without the intermediate formation of an amide. Kobayashi et al. (Tetrahedron 46:5587-5590 (1990); J. Bacteriology 172:4807-4815 (1990)) have described an aliphatic nitrilase isolated from Rhodococcus rhodochrous K22 which catalyzed the hydrolysis of a variety of aliphatic nitrites to the corresponding carboxylic acids. A nitrilase from Comamonas testosteroni has been isolated that can convert a range of aliphatic xcex1,xcfx89-dinitriles to either the corresponding xcfx89-cyanocarboxylic acids or dicarboxylic acids (CA 2,103,616; Lxc3xa9vy-Schil et al., Gene 161:15-20 (1995)). Aliphatic nitrilases are also produced by Rhodococcus rhodochrous NIMBI 11216 (Bengis-Garber et al., Appl. Microbiol. Biotechnol. 32:11-16 (1989); Gradley et al., Biotechnology Lett. 16:41-46 (1994)), Rhodococcus rhodochrous PA-34 (Bhalla et al., Appl. Microbiol. Biotechnol. 37:184-190 (1992)), Fusarium oxysporum f. sp. melonis (Goldlust et al., Biotechnol. Appl. Biochem. 11:581-601 (1989)), Acinetobacter sp. AK 226 (Yamamoto et al., Agric. Biol. Chem. 55:1459-1473 (1991)), Alcaligenes faecalis ATCC 8750 (Yamamoto et al., J. Ferment. Bioeng. 73:425-430 (1992)), and Acidovorax facilis 72W (Gavagan et al., J. Org. Chem. 63:4792-4801 (1998)).
The problem to be solved, therefore, is to provide new catalysts useful for converting nitrites to their corresponding carboxylic acids at high yield. More specifically, the ability to convert a nitrile functional group in a compound to the corresponding carboxylic acid in the presence of a hydroxyl group that can undergo elimination would be extremely useful.
A process is disclosed for hydrolyzing 3-hydroxynitrile to 3-hydroxycarboxylic acid. The process includes the steps of (a) contacting a 3-hydroxynitrile in an aqueous reaction mixture with an enzyme catalyst characterized by 1) nitrile hydratase and amidase activity or 2) nitrilase activity; and (b) optionally, recovering the 3-hydroxycarboxylic acid produced in step (a). More particularly, 3-hydroxyvaleronitrile is converted in the invention to 3-hydroxyvaleric acid in high yield at up to 100% conversion, using as an enzyme catalyst 1) nitrile hydratase activity and amidase activity or 2) nitrilase activity of a microbial cell.
Further embodiments of the invention to hydrolyze 3-hydroxynitrile to 3-hydroxycarboxylic acid use an enzyme catalyst having 1) nitrile hydratase activity and amidase activity or 2) nitrilase in the form of whole microbial cells, permeabilized microbial cells, one or more cell components of a microbial cell extract, partially-purified enzyme(s), or purified enzyme(s). Preferably, the form of enzyme catalyst is whole microbial cells, or (for the embodiment of the process using nitrile hydratase and amidase activity) an additional preferred form of enzyme catalyst is as partially purified or purified enzyme. These different forms of enzyme catalyst can be immobilized on or in a soluble or insoluble support. Microorganisms characterized by nitrile hydratase activity and amidase activity and useful in the process are Acidovorax facilis 72W (ATCC 55746), Comamonas testosteroni 22-1(ATCC PTA-1853), Comamonas testosteroni 5-MGAM-4D (ATCC 55744), Dietzia sp. ADL1 (ATCC PTA-1854), Syctalidium spp. 3LD-122P (ATCC PTA-1855), Rhodococcus sp. 25-1 (ATCC PTA-1856), and Pseudomonas putida 5B-MGN-2P (NRRL-B-18668). Microorganisms characterized by a nitrilase activity and useful in the process are Acidovorax facilis 72W (ATCC 55746) (after heating at 50xc2x0 C. for 0.5-1 hour to inactivate undesirable nitrile hydratase and amidase activities), Acidovorax facilis 72-PF-17 (ATCC 55745) and Acidovorax facilis 72-PF-15 (ATCC 55747).
The invention is useful in producing a highly branched copolyester comprising at least two repeat units derived from at least one linear 3-hydroxycarboxylic acid or its ester of the structure R2Oxe2x80x94CR4R5CR6R7C(O)OR1 and at least one hyperbranching hydroxycarboxylic acid or its ester of the structure (R2O)n-R-[C(O)OR1]m, wherein R is C1-12 hydrocarbyl radical with n+m free valencies, R1 is H, C1-12 or hydroxyl substituted C1-12 hydrocarbyl radical, R3, R4, R5, R6, R7 is H or C1-12 hydrocarbyl radical, R2 is H or (O)CR3, n+m is 3 or more, and provided that one of n and m is 1.
Additionally, the invention includes a process for synthesizing a highly branched copolyester product comprising the steps of: (a) contacting and heating a mixture of (1) at least one hyperbranching hydroxycarboxylic acid or its ester of the structure (R2O)n-R-[C(O)O R1]m, wherein R is C1-12 hydrocarbyl radical with n+m free valencies, R1 is H, C1-12 or hydroxyl substituted C1-12 hydrocarbyl radical, R3, R4, R5, R6, R7 is H or C1-12 hydrocarbyl radical, R2 is H or (O)CR3, n+m is 3 or more, and provided that one of n and m is 1, (2) a linear 3-hydroxycarboxylic acid or its ester of the structure R2Oxe2x80x94CR4R5CR6R7C(O)OR1, and (3) an esterification catalyst; and (b) collecting the highly branched copolyester product of step (a). In this process, the hyperbranching hydroxycarboxylic acid is preferably dimethylolpropionic acid or trimethylolacetic acid, the linear hydroxycarboxylic acid is preferably 3-hydroxyvaleric acid. Examples of the linear 3-hydroxycarboxylic acids include but are not limited to the following compounds: 3-hydroxypropionic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxy-3-isopropyl-4-methylpentanoic acid, 3-hydroxy-3-phenylpropanoic acid, 2-propyl-3-hydroxypentanoic acid, 3-hydroxy-3-methyl-n-valeric acid, and 3-hydroxy-2,2-dimethylpropionic acid. The esterification catalyst may be any conventionally known such as a protonic acid, Lewis acid, or a basic catalyst including sulfonic acids, phosphoric and phosphonic acids, titanium alkoxides, dialkyltin oxide, oxides, carbonates and carboxylates of tin, zinc, manganese, calcium, magnesium, or antimony. The esterification catalyst is preferably tin dicarboxylate or a protonic acid.
An additional embodiment of the invention uses a linear 3-hydroxycarboxylic acid produced with an enzyme catalyst as described herein to synthesize a highly branched copolyester product. The enzymatically-produced linear 3-hydroxycarboxylic acid is heated in contact with at least one hyperbranched hydroxycarboxylic acid or its ester as discussed herein, and an esterification catalyst.
Applicants have made the following biological deposits under the terms of the Budapest Treaty:
As used herein, xe2x80x9cATCCxe2x80x9d refers to the American Type Culture Collection international depository located at 10801 University Boulevard, Manassas, Va. 20110-2209 U.S.A. As used herein, xe2x80x9cNRRLxe2x80x9d refers to the Agricultural Research Service Culture Collection, part of the Microbial Properties Research Unit located at the National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Ill. 61604, U.S.A. The xe2x80x9cInt""l Depository Designation No.xe2x80x9d is the accession number to cultures on deposit with the ATCC or the NRRL, respectively.
The listed deposit(s) will be maintained in the indicated international depository for at least thirty (30) years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.
Applicants have solved the stated problem by using an enzyme catalyst to convert the nitrile functional group of a 3-hydroxynitrile to its corresponding carboxylic acid in the presence of a hydroxyl group that can undergo elimination. This process offers significant advantages in the preparation of, for example, 3-hydroxyvaleric acid or 3-hydroxypropionic acid over other chemical or enzymatic methods of nitrile hydrolysis, and makes possible the preparation of 3-hydroxycarboxylic acids such as 3-hydroxyvaleric acid or 3-hydroxypropionic acid in high yield from relatively inexpensive and readily prepared starting materials, with very little byproduct and waste production.
The invention relates to the production of improved polyesters for use primarily in coatings, but also in fibers, films, and moldings. Material of the invention also has utility as a crosslinker. The 3-hydroxyvaleric acid produced by the present invention is useful as an ingredient in the preparation particularly of highly branched polyesters in combination with the hyperbranching hydroxycarboxylic comonomer and is useful as a (co)monomer in biodegradable polyester production.
Specifically, a process to prepare 3-hydroxyvaleric acid from 3-hydroxyvaleronitrile in high yields has been demonstrated that uses 1) nitrilase activity, or 2) nitrile hydratase activity and amidase activity of microbial cells. A nitrilase enzyme directly converts an aliphatic or aromatic nitrile to the corresponding carboxylic acid, without the formation of the corresponding amide intermediate (Equation 1), whereas nitrile hydratase (NHase) initially converts an aliphatic or aromatic nitrile to an amide, and then the amide is subsequently converted by the amidase to the corresponding carboxylic acid (Equation 2): 
Similar yields of 3-hydroxyvaleric acid have been obtained using purified enzyme(s), cell extracts, microbial cells and immobilized microbial cells, as described in the accompanying Examples.
Several classes of highly branched copolyester polyols have been prepared using dimethylolpropionic acid as a branching comonomer and a variety of linear hydroxycarboxylic acids and lactones. Some of these polymers demonstrate attractive characteristics. The corresponding block copolymers with similar overall composition but of different microstructure have been reported (e.g., DMPA/xcex5-caprolactone block copolymers described in Macromolecules, 30: 8508 (1997) and J. Polym. Sci. Part (A): Polymer Chemistry 36: 2793 (1998)). Highly branched copolyester polyol substrates for reactive coatings with desirable, significantly enhanced Tg have now been obtained with the present invention when 3-hydroxycarboxylic acids such as 3-hydroxyvaleric acid or 3-hydroxy-2,2-dimethylpropionic acid were substituted for xcex5-caprolactone as a linear comonomer in the copolymerization with dimethylolpropionic acid or trimethylolacetic acid. The higher Tg signficantly expands the range of applications to which branched copolyesters can be put.
The claimed invention for preparing 3-hydroxycarboxylic acids generates little waste or reaction byproducts, and the 3-hydroxycarboxylic acid is readily recovered from the product mixture. Previously known chemical methods for the hydrolysis of 3-hydroxynitriles cannot produce the high yields and selectivities to 3-hydroxycarboxylic acids obtained using enzyme-catalyzed nitrile hydrolysis. Non-enzymatic nitrile hydrolysis reactions typically involve heating solutions of the nitrile at elevated temperatures, often times in the presence of strong acid or base, while the enzyme-catalyzed reactions described above are carried out at ambient temperature in aqueous solution and at neutral pH with no added acid or base. For example, aqueous barium hydroxide has been used to hydrolyze 3-aminopropionitrile to 3-alanine in 85 to 90% yield (Ford, Org. Synth., Coll. vol. III: 34-36 (1955)), and 3-cyanobutyric acid to methylsuccinic acid in 72% yield (Brown, Org Synth., Coll. vol. III: 615-617 (1955)); repeating the first of these two procedures with 3-hydroxyvaleronitrile produced little or no detectable 3-hydroxyvaleric acid (see Comparative Example).
In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.
xe2x80x9cNitrile hydratasexe2x80x9d is abbreviated NHase.
xe2x80x9cNitrile hydratase-deficientxe2x80x9d describes cells that have no nitrile hydratase activity as a result of heat treatment.
xe2x80x9cEnzyme catalystxe2x80x9d refers to a catalyst which is characterized by 1) nitrilase activity or 2) nitrile hydratase activity and amidase activity. The enzyme catalyst may be in the form of a whole microbial cell, permeabilized microbial cell(s), one or more cell components of a microbial cell extract, partially purified enzyme, or purified enzyme.
A saturated xe2x80x9chydrocarbyl radicalxe2x80x9d is defined as any radical composed exclusively of carbon and hydrogen, where single bonds are exclusively used to join carbon atoms together. Thus, any stable arrangement of carbon and hydrogen atoms, having at least one carbon atom, is included within the scope of a saturated hydrocarbon radical.
The terms xe2x80x9chyperbranchedxe2x80x9d, xe2x80x9chighly branchedxe2x80x9d, and xe2x80x9cdendritic macromoleculesxe2x80x9d (dendrimers) can generally be described as three-dimensional, highly branched molecules having a tree-like structure. Dendrimers are highly symmetrical, while similar macromolecules designated as hyperbranched or highly branched may to a certain degree hold an asymmetry, yet maintain the highly branched tree-like structure. Dendrimers can be said to be monodisperse variations of hyperbranched macromolecules. Hyperbranched, highly branched, and dendritic macromolecules normally consist of an initiator or nucleus having one or more reactive sites and a number of surrounding branching layers and, optionally, a layer of chain terminating molecules. The layers are usually called generations.
xe2x80x9c3-Hydroxynitrilexe2x80x9d is equivalent to xe2x80x9cxcex2-Hydroxynitrilexe2x80x9d. 3-Hydroxynitriles include but are not limited to the following compounds: 3-hydroxypropionitrile, 3-hydroxybutyronitrile, 3-hydroxyvaleronitrile, 3-hydroxyhexanenitrile, 3-hydroxyheptanenitrile, 3-hydroxyoctanenitrile, 3-hydroxynonanenitrile, 3-hydroxy-3-isopropyl-4-methylpentanenitrile, 3-hydroxy-3-phenylpropanenitrile, 2-propyl-3-hydroxypentanenitrile and 3-hydroxy-3-methyl-n-pentanenitrile.
xe2x80x9c3-Hydroxycarboxylic acidxe2x80x9d is equivalent to xe2x80x9cxcex2-Hydroxycarboxylic acidxe2x80x9d. 3-Hydroxycarboxylic acids include but are not limited to the following compounds: 3-hydroxypropionic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxy-3-isopropyl-4-methylpentanoic acid, 3-hydroxy-3-phenylpropanoic acid, 2-propyl-3-hydroxypentanoic acid, 3-hydroxy-2,2-dimethylpropionic acid, and 3-hydroxy-3-methyl-n-valeric acid.
xe2x80x9c3-Hydroxyvaleronitrilexe2x80x9d is also known as 3-hydroxypentanenitrile and xcex2-hydroxyvaleronitrile.
xe2x80x9c3-Hydroxyvaleric acidxe2x80x9d is also known as 3-hydroxypentanoic acid and xcex2-hydroxyvaleric acid.
xe2x80x9c3-Hydroxypropionitrilexe2x80x9d is also known as hydracrylonitrile, 3-cyanoethanol, 3-hydroxyethyl cyanide, 3-hydroxypropionitrile, 1-cyano-2-hydroxyethane, 2-cyanoethanol, 2-cyanoethyl alcohol, 2-hydroxycyanoethane, 2-hydroxyethyl cyanide, 3-hydroxypropanenitrile, 3-hydroxypropionitrile, ethylene cyanohydrin, glycol cyanohydrin.
xe2x80x9c3-Hydroxypropionic acidxe2x80x9d is also known as hydracrylic acid, xcex2-hydroxypropionic acid, 3-lactic acid, 2-deoxyglyceric acid, 3-hydroyxypropanoic acid, and ethylenelactic acid.
xe2x80x9cDimethylolpropionic acidxe2x80x9d is also known as 2,2-bis(hydroxymethyl)-propionic acid and xcex1,xcex1-bis(hydroxymethyl)propionic acid.
xe2x80x9cTrimethylolacetic acidxe2x80x9d is also known as 3-hydroxy-2,2-bis(hydropoxymethyl)-propanoic acid, 2,2-bis(hydroxymethyl)-hydracrylic acid, 2-carboxy-2-(hydroxymethyl)-1,3-propanediol; 3-hydroxy-2,2-bis(hydroxymethyl)Propionic acid; 3-hydroxy-2,2 -dihydroxymethylpropionic acid; tris(hydroxymethyl)acetic acid.
The terms xe2x80x9cprotic acidxe2x80x9d and xe2x80x9cprotonic acidxe2x80x9d refer to acids having an ionizable proton (i.e., capable of acting as a proton donor) strongly or weakly acidic. These acids include, but are not limited to, aromatic or aliphatic carboxylic acids, aromatic or aliphatic sulfonic acids, phosphoric acid, sulfuric acid, sulfurous acid, nitric acid, perchloric acid, hydrochloric acid, and the like (often referred to as Lowry-Brxc3x8nsted acids). Examples of non-protonic acids (i.e., acids that can accept an electron pair to form a covalent bond) are Lewis acids such as boron trifluoride, aluminum trichloride, and stannic chloride.
Growth of Microbial Enzyme Catalysts:
Microbial strains used for conversion of 3-hydroxynitriles were isolated as described below. Frozen 15% glycerol stocks were maintained at xe2x88x9265xc2x0 C. to xe2x88x9270xc2x0 C.
Rhodococcus sp. 25-1 Comamonas testosteroni 22-1, Acidovorax facilis 72W, and Comamonas testosteroni 5-MGAM-4D were enriched from soil collected in Orange, Tex., U.S.A., using standard enrichment procedures with E2 basal medium (Table 1) (pH 7.2).
Table 2 contains modifications that were made to the E2 basal medium for the enrichments described above.
xe2x80x9cComamonas testosteroni 5-MGAM-4D 1% methylglutaramide 0.6% glycolxe2x80x9d
Scytalidium spp. 3LD-122P and Pseudomonas putida 5B-MGN-2P were enriched from soil collected in Orange, Tex., U.S.A., using standard enrichment procedures with PR basal medium (Table 3) (pH 7.2).
Table 4 contains modifications that were made to the PR basal medium for the enrichments described above.
Dietzia sp. ADL1 was isolated from an enrichment culture. The enrichment culture was established by inoculating 1 mL of sludge into 10 mL of S12-N medium in a 50 mL screw cap Erlenmeyer flask. S12-N medium contains the following: Na2SO4, 10 mM; potassium phosphate buffer, pH 7.0, 50 mM; MgCl2, 2 mM; CaCl2, 0.7 mM; MnCl2, 50 xcexcM; FeCl3, 1 xcexcM; ZnCl3, 1 xcexcM; CuSO4, 1.72 xcexcM; CoCl2, 2.53 xcexcM; Na2MoO2, 2.42 xcexcM; FeSO4, 0.0001%; yeast extract, 0.001%; and thiamine hydrochloride, 2 xcexcM. The sludge was obtained from a waste water treatment testing system used by E. I. du Pont de Nemours and Company in Victoria, Tex. The enrichment culture was supplemented with 100 ppm adiponitrile added directly to the culture medium and was incubated at 30xc2x0 C. with reciprocal shaking. The enrichment culture was maintained by adding 100 ppm of toluene every 2-3 days. The culture was diluted every 10 days by replacing 9 mL of the culture with the same volume of S12-N medium. Bacteria that utilize adiponitrile as a sole source of carbon, nitrogen, and energy were isolated by spreading samples of the enrichment culture onto S12-N agar (S12-N medium with 1.5% Difco Noble Agar). Adiponitrile (10 xcexcL) was placed on the interior of each Petri dish lid. The Petri dishes were sealed with parafilm and incubated upside down at 28xc2x0 C. Representative bacterial colonies were then single colony passaged several times on S12-N agar with adiponitrile supplied on the interior of each Petri dish lid. The Petri dishes were sealed with parafilm and incubated upside down at 28xc2x0 C. Dietzia sp. ADL1 was one of the several strains isolated.
The various strains were grown aerobically under the following conditions (Tables 5 and 6) for testing nitrile transformation activity.
Additionally, Acidovorax facilis 72W was grown aerobically. At inoculation, the fermenter contained 8.5 L of Fermenter Medium (Table 6) plus 218 g of Nutrient Feed solution (see below), giving a starting concentration of approximately 7 g/L glycerol. Dissolved oxygen was held at 25% of saturation, at 32xc2x0 C., and pH at 6.8-7.0. At 18 h post inoculation, feeding of Nutrient Feed solution began. The Nutrient Feed solution included the following components which were sterilized separately and combined after cooling: potassium phosphate, monobasic, 19.6 g in 0.25 L deionized water; magnesium sulfate, heptahydrate, 3.3 g plus sulfuric acid, 4 mL, in 0.15 L deionized water; Trace Metal (Table 6) solution, 67 mL, plus 400 g glycerol in 0.80 L deionized water. Initially, the Nutrient Feed solution was added at a rate of 0.4 g feed/minute (0.15 g glycerol/min). At 26 h, the feed rate was increased to 0.9 g feed/min (0.3 g glycerol/min). A final increase in feed rate to 1.8 g feed/min (0.6 g glycerol/min) was made at 34 h. 72W Cells were harvested at 58 hours.
Harvested cells were frozen at xe2x88x9265 to xe2x88x9270xc2x0 C. until used for nitrile transformation. For use as an enzyme catalyst having only nitrilase activity, a 10 to 50% (wet cell weight) suspension of Acidovorax facilis 72W cells in 0.35 M phosphate buffer (pH 7.0) were first heated to 50xc2x0 C. for 1 h to inactivate the nitrile hydratase and amidase enzymes present without measurably decreasing the nitrilase activity. Acidovorax facilis 72W cells which were not heat-treated at 50xc2x0 C., and which had nitrilase, and nitrile hydratase and amidase activities produced yields of 3-hydroxyvaleric acid similar to heat-treated, nitrilase-only containing cells.
Two mutants of the Acidovorax facilis 72W (ATCC 55746) strain have been prepared (U.S. Pat. No. 5,858,736, incorporated by reference) which produce only very low levels of the undesirable nitrile hydratase activity responsible for non-regioselective nitrile hydrolysis of aliphatic dinitriles. These nitrile hydratase-deficient mutant strains, Acidovorax facilis 72-PF-15 (ATCC 55747) and Acidovorax facilis 72-PF-17 (ATCC 55745), do not require heat-treatment of the cells prior to use as an enzyme catalyst for the hydrolysis of 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid. In cases where the regioselectivity of the nitrilase is not required, the Acidovorax facilis 72W (ATCC 55746) strain does not have to be heat-treated in order to deactivate the non-regioselective nitrile hydratase activity.
Preparation of Cell Extract:
All steps in this procedure were performed at 5xc2x0 C. and at pH 7.5. A 25 wt % suspension of Comamonas testosteroni 22-1 (ATCC PTA-1853) wet cell paste was prepared in 100 mM potassium phosphate buffer (pH 7.0), 0.1 mM phenyl methyl sulfonyl fluoride (PMSF) and 2.0 mM dithiothreitol. An extract of this suspension was prepared by passage through a French press (American Instrument Co., Silver Springs, Md., U.S.A.) according to methods known to the art. The cell extract was prepared by a centrifugation at 27,500 g for 30 minutes to remove cell debris.
Preparation of 3-Hydroxyvaleronitrile:
3-Hydroxyvaleronitrile has been prepared by reacting hydrogen cyanide with 1,2-epoxybutane in the presence of triethylaluminum (FR 1446127), and by the reaction of acetonitrile and propionaldehyde in the presence of di-n-butylboryl triflate (Hamana et al., Chem. Lett. 1401-1404 (1982)). Optically active 3-hydroxyvaleronitrile has been prepared by the lipase-catalyzed hydrolysis of 2-cyano-1-methylethyl acetate (Itoh et al., J. Org. Chem. 62:9165-9172 (1997)).
Hydrolysis of 3-Hydroxynitrile to 3-Hydroxycarboxylic Acid:
The hydrolysis reaction is performed by mixing a 3-hydroxynitrile, for example, 3-hydroxyvaleronitrile, with an aqueous suspension of the appropriate enzyme catalyst. Whole microbial cells can be used as an enzyme catalyst without any pretreatment. Alternatively, they can be immobilized in a polymer matrix (e.g., alginate beads or polyacrylamide gel (PAG) particles) or on an insoluble solid support (e.g., celite) to facilitate recovery and reuse of the enzyme catalyst. The enzyme(s) can also be isolated from the whole cells and used directly as a catalyst, or the enzyme(s) can be immobilized in a polymer matrix or on an insoluble support. Methods for the immobilization of cells, or the isolated enzymes, have been widely reported and are well known to those skilled in the art (Methods in Biotechnology, Vol. 1: Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, N.J., USA; 1997).
The concentration of enzyme catalyst in the aqueous reaction mixture depends on the specific catalytic activity of the enzyme catalyst and is chosen to obtain the desired rate of reaction. The wet cell weight of the microbial cells used as catalyst in hydrolysis reactions typically ranges from 0.001 grams to 0.100 grams of wet cells per mL of total reaction volume, preferably from 0.002 grams to 0.050 grams of wet cells per mL. The specific activity of the microbial cells (IU/gram wet cell wt.) is determined by measuring the rate of conversion of a 0.10 M solution of 3-hydroxyaleronitrile to 3-hydroxyvaleric acid at 25xc2x0 C., using a known weight of microbial cell catalyst. An IU (International Unit) of enzyme activity is defined as the amount of enzyme activity required to convert one micromole of substrate to product per minute.
The temperature of the hydrolysis reaction is chosen to optimize both the reaction rate and the stability of the enzyme catalyst activity. The temperature of the reaction may range from just above the freezing point of the suspension (approximately 0xc2x0 C.) to 65xc2x0 C., with a preferred range of reaction temperature of from 5xc2x0 C. to 35xc2x0 C. The microbial cell catalyst suspension may be prepared by suspending the cells in distilled water, or in a aqueous solution of a buffer which will maintain the initial pH of the reaction between 5.0 and 10.0, preferably between 6.0 and 9.0. As the reaction proceeds, the pH of the reaction mixture may change due to the formation of an ammonium salt of the carboxylic acid from the corresponding nitrile functionality. The reaction can be run to complete conversion of 3-hydroxynitrile with no pH control, or a suitable acid or base can be added over the course of the reaction to maintain the desired pH.
3-Hydroxyvaleronitrile was found to be completely miscible with water in all proportions at 25xc2x0 C. In cases where reaction conditions are chosen such that the solubility of 3-hydroxyvaleronitrile is also dependent on the temperature of the solution and/or the salt concentration (buffer or product 3-hydroxyvaleric acid ammonium salt) in the aqueous phase, the reaction mixture may initially be composed of two phases: an aqueous phase containing the enzyme catalyst and dissolved 3-hydroxyvaleronitrile, and an organic phase (the undissolved 3-hydroxyvaleronitrile). As the reaction progresses, the 3-hydroxyvaleronitrile dissolves into the aqueous phase, and eventually a single phase product mixture is obtained. The reaction may also be run by adding the 3-hydroxyvaleronitrile to the reaction mixture at a rate approximately equal to the enzymatic hydrolysis reaction rate, thereby maintaining a single-phase aqueous reaction mixture, and avoiding the potential problem of substrate inhibition of the enzyme at high starting material concentrations.
3-Hydroxyvaleric acid may exist in the product mixture as a mixture of the protonated carboxylic acid and its corresponding ammonium salt (dependent on the pH of the product mixture), and may additionally be present as a salt of the carboxylic acid with any buffer which may additionally be present in the product mixture. The 3-hydroxyvaleric acid product may be isolated from the reaction mixture as the protonated carboxylic acid, or as a salt of the carboxylic acid, as desired.
The final concentration of 3-hydroxyvaleric acid in the product mixture at complete conversion of 3-hydroxyvaleronitrile may range from 0.001 M to the solubility limit of the 3-hydroxyvaleric acid product. Preferably, the concentration of 3-hydroxyvaleric acid will range from 0.10 M to 2.0 M. 3-Hydroxyvaleric acid may be isolated from the product mixture (after removal of the catalyst) by adjusting the pH of the reaction mixture to between 1.0 and 2.5 with concentrated hydrochloric acid, saturation of the resulting solution with sodium chloride, and extraction of 3-hydroxyvaleric acid with a suitable organic solvent such as methyl t-butyl ether, ethyl ether, or dichloromethane. The combined organic extracts are then combined, stirred with a suitable drying agent (e.g., magnesium sulfate), filtered, and the solvent removed (e.g., by rotary evaporation) to produce the desired product in high yield and in high purity (typically 98-99% pure). If desired, the product can be further purified by recrystallization or distillation.
The enzymatic hydrolysis of 3-hydroxypropionitrile to 3-hydroxypropionic acid was performed using methods similar to those described above for 3-hydroxyvaleronitrile (see accompanying Examples), and produced 3-hydroxypropionic acid in 99% to 100% yields at complete conversion of 3-hydroxypropionitrile. Additional 3-hydroxynitriles which may be converted by the present methods to the corresponding 3-hydroxycarboxylic acids include, but are not limited to, 3-hydroxybutyronitrile, 3-hydroxyhexanenitrile, 3-hydroxyheptanenitrile, 3-hydroxyoctanenitrile, 3-hydroxynonanenitrile, 3-hydroxy-3-isopropyl-4-methylpentanenitrile, 3-hydroxy-3-phenylpropanenitrile, 2-propyl-3-hydroxypentanenitrile and 3-hydroxy-3-methyl-n-pentanenitrile. When the 3-hydroxynitrile (or its hydrolysis products) is not completely water miscible, the reaction is run in a two-phase, aqueous/organic reaction mixture as described above, using methods known to those skilled in the art.
In all of the polymerizations described herein to make highly branched copolyesters, wherein at least two repeat units are derived from at least one linear 3-hydroxycarboxylic acid or its ester of the structure R1Oxe2x80x94CR4R5CR6R7C(O)OR1 and at least one hyperbranching hydroxycarboxylic acid or its ester of the structure (R2O)n-R-[C(O)OR1]m, wherein R is C1-12 hydrocarbyl radical with n+m free valencies, R1 is H, C1-12 or hydroxyl substituted C1-12 hydrocarbyl radical, R3, R4, R5, R6, R7 is H or C1-12 hydrocarbyl radical, R2 is H or (O)CR3, n+m is 3 or more, and provided that one of n and m is 1, these repeat units may also be derived from equivalent compounds that will form polyesters, such as esters of the hydroxycarboxylic acids. The compound (R2O)n-R-[C(O)ORxe2x80x2]m, by virtue of being a tri- or higher functional, is sometimes called a hyperbranching monomer. More than one such monomer may be present in such a polymerization. It is preferred that n+m is three or four. Normal esterification catalysts well known in the art may be used with these monomers to form polyesters (for example, a protonic acid, Lewis acid, or a basic catalyst including sulfonic acids, phosphoric and phosphonic acids, titanium alkoxides, dialkyltin oxide, oxides, carbonates and carboxylates of tin, zinc, manganese, calcium, magnesium, or antimony). Methods for making polyesters are well known in the art.
In the following examples, which serve to further illustrate the invention and not to limit it, the % recovery of 3-hydroxynitrile and the % yields of 3-hydroxycarboxylic acid and 3-hydroxycarboxylic acid amide were based on the initial amount of 3-hydroxynitrile present in the reaction mixture. This data was determined by HPLC using a refractive index detector and either a Supelcosil LC-18-DB column (15 cmxc3x974.6 mm diameter) with 7.5% (v/v) methanol in aqueous 10 mM acetic acid/10 mM sodium acetate as mobile phase (for 3-hydroxyvaleronitrile reactions), or a Bio-Rad HPX-87H column (30 cmxc3x977.8 mm diameter) with 0.01 N sulfuric acid as mobile phase (for 3-hydroxypropionitrile reactions). The isolated yields of 3-hydroxyvaleric acid reported in the following examples were not optimized for complete recovery of the product.